Universidade Estadual De Feira De Santana Departamento De Ciências Biológicas Programa De Pós Graduação Em Botânica

UNIVERSIDADE ESTADUAL DE FEIRA DE SANTANA DEPARTAMENTO DE CIÊNCIAS BIOLÓGICAS PROGRAMA DE PÓS GRADUAÇÃO EM BOTÂNICA

DIVERSIDADE FILOGENÉTICA DE APOCYNACEAE NO NORDESTE E

SUAS IMPLICAÇÕES PARA A CONSERVAÇÃO DA BIODIVERSIDADE

LARA PUGLIESI DE MATOS

Dissertação apresentada ao Programa de Pós- Graduação em Botânica da Universidade Estadual de Feira de Santana como parte dos requisitos para obtenção do título de Mestre em Botânica.

ORIENTADOR: PROF. Dr. ALESSANDRO RAPINI

Feira de Santana-BA

2014

BANCA EXAMINADORA

PROF. DR.ª PATRÍCIA LUZ RIBEIRO

UNIVERSIDADE FEDERAL DO RECÔNCAVO DA BAHIA - UFRB

PROF. DR. LUCIANO PAGANUCCI DE QUEIROZ

UNIVERSIDADE ESTADUAL DE FEIRA DE SANTANA - UEFS

PROF. DR. ALESSANDRO RAPINI

UNIVERSIDADE ESTADUAL DE FEIRA DE SANTANA - UEFS

ORIENTADOR E PRESIDENTE DA BANCA

Feira de Santana-BA

2014

―O JOVEM QUE PRETENDE SER CIENTISTA

DEVE ESTAR DISPOSTO A ERRAR 99 VEZES

ANTES DE ACERTAR UMA...‖

CHARLES KETTERING

SUMÁRIO

AGRADECIMENTOS ...... 1

APRESENTAÇÃO ...... 4

TROPICAL REFUGES WITH EXCEPTIONALLY HIGH PHYLOGENETIC DIVERSITY REVEAL CONTRASTING PHYLOGENETIC STRUCTURES ...... 5

SUPPLEMENTARY FIGURES ...... 28

APPENDICES ...... 30

AGRADECIMENTOS

Em primeiro lugar agradeço a Deus. Acredito que em cada momento de dificuldade meus passos foram guiados por ele, me conduzindo até aqui.

Ao Programa de Pós-graduação em Botânica (PPGBot - UEFS) por toda estrutura e apoio financeiro fornecidos.

Ao Cnpq pelo apoio concedido através da bolsa de mestrado.

A meu orientador Alessandro Rapini, por ter aceitado meu convite de orientação, pela sua competência, comprometimento e dedicação, buscando sempre resultados de excelência, e esperando sempre o melhor de seus orientados.

Ao professor Luciano Paganucci de Queiroz por ter aceitado o convite de participação desta banca avaliadora mesmo com tantos outros compromissos, pelas ótimas contribuições, questionamentos e sugestões feitas na avaliação deste trabalho, e ainda pelos ensinamentos transmitidos durante as disciplinas tão bem ministradas. Sou muito grata.

À professora Patrícia Luz Ribeiro também por aceitar o convite para compor esta banca avaliadora e por suas contribuições na melhoria desta trabalho, e pelas discussões e ensinamentos sobre o BEAST, e por sempre estar disposta a ajudar e compartilhar sua experiência com quem está começando.

Aos professores Cássio Van den Berg e Abel Conceição por dividirem seu conhecimento e estarem sempre abertos a discussões e por suprirem minhas dúvidas constantes sobre filogenia, estatística e análises de similaridade.

Aos professores Flávio França e Efigênia de Melo por serem sempre tão prestativos e receptivos a qualquer estudante do mundo da botânica, tendo sempre um espacinho no

Laboratório de Taxonomia Vegetal, e por me abrigarem durante muito tempo da iniciação científica.

A Adriana pela boa vontade e simpatia constantes em lidar com as situações diárias muitas vezes estressantes na secretaria do Programa de pós-graduação.

Aos funcionários da portaria e da limpeza, pela simpatia e por tornarem o LABIO um ambiente sempre agradável para desenvolvermos nossos trabalhos.

À minha família...

Agradeço aos meus pais Sérgio e Márcia por todo amor e dedicação à nossa família, por todo apoio em todos os momentos deste mestrado, sempre com bons conselhos e me dando muita força para prosseguir, vocês são a base que me sustenta.

A meu irmão Sérgio pelo companheirismo, pelo apoio e força nos momentos mais difíceis deste mestrado.

Aos meus amigos...

À Cássia Bitencourt, por todos os conselhos, força e amizade, todo conhecimento compartilhado, pela ajuda em todos os momentos, não importando hora nem lugar, você sempre esteve disponível, e foi fundamental para que eu conseguisse concluir esta dissertação.

A meu namorado Dudu (Eduardo Menezes), por cada palavra de carinho, de compreensão e incentivo, mesmo nos momentos mais estressantes, toda vez que por algum motivo me desanimei, por tentar até ser um pouco biólogo em alguns momentos para me incentivar. Foi fundamental estar com você neste momento.

A Kamilla Lopes minha amiga e companheira de dificuldades, por dividir mais uma etapa acadêmica, juntas, sempre me apoiando e fortalecendo. A Silvana Santos e Luiz por

2 toda força e apoio, sempre se mostrando disponíveis a ajudar, pelas palavras de incentivo em qualquer momento de dificuldade. Sem o apoio de vocês três foi imprescindível.

À Pricila Lopes e Danielle Mendes, minhas amigas desde à graduação, por sempre me darem apoio e incentivo.

Aos colegas e amigos de pós-graduação que sempre torceram, se disponibilizaram a ajudar, com palavras de incentivo e otimismo: Danilo José, Gabriela Barros, Evelyne

Marques, Earl Chagas, Amanda Priscila, Denis Carvalho, Fábio Espírito Santo, Moabe,

Ariane, Cris Isnac, Christian, Bernarda, Lamarck, Patrícia, Pétala.

A Thiago Araújo pela prestatividade e ajuda no uso das ferramentas estatísticas e do software R.

A Leilton Damascena por compartilhar seus conhecimentos sobre o ArcGis, quando eu nem fazia ideia de como começar.

A Naron por sua ajuda também no ArcGis, pela sua prestatividade e simpatia em ajudar as pessoas.

À minha primeira orientadora no mundo da botânica, Maria José Gomes de Andrade, por todos os ensinamentos profissionais, mas também pelo exemplo de ética que é no âmbito cientifico.

A todos os colegas do grupo de discussão dos orientados de Rapini, Ana Luisa Côrtes,

Uiara Catharina, Hibert, por sempre que possível compartilharem informações e conhecimentos.

A todos vocês serei eternamente grata.

APRESENTAÇÃO

Diante da acelerada perda de biodiversidade, causada entre outros fatores pela perda de hábitat, compreendemos a urgente necessidade de reunir os conhecimentos científicos acumulados, ainda que incompletos, e contribuir para a identificação e conservação de áreas de alta biodiversidade que abriguem importantes processos evolutivos e ecológicos. Este trabalho integra duas fontes principais de dados: a distribuição de espécies de Apocynaceae do nordeste brasileiro e sequências de DNA para suas cinco subfamílias. Através destes dados utilizamos a diversidade filogenética (PD, Phylogenetic Diversity) mediante sua estrutura em metacomunidades para avaliar a distribuição da diversidade evolutiva na

Caatinga e Mata Atlântica do nordeste brasileiro. Estes parâmetros conseguem mensurar intrinsecamente aspectos evolutivos e funcionais da biodiversidade, ampliando o alcance da avaliação de áreas para conservação.

O mapeamento da PD no Nordeste revelou duas áreas que abrigam os maiores valores de diversidade filogenética: uma inserida na Caatinga, os Campos Rupestres, na Chapada

Diamantina, e outra na Mata Atlântica, a ecorregião das florestas costeiras da Bahia, as quais se tornaram nosso foco de investigação. Nossos dados revelaram estruturas filogenéticas diferentes entre as metacomunidades dessas duas regiões. Esses resultados permitiram inferir histórias evolutivas e indicar potenciais estratégias de conservação para cada uma delas.

ARTIGO

TROPICAL REFUGES WITH EXCEPTIONALLY HIGH

PHYLOGENETIC DIVERSITY REVEAL CONTRASTING

PHYLOGENETIC STRUCTURES

Artigo publicado em International Journal of Biodiversity

http://www.hindawi.com/journals/ijbd/2015/758019/

ABSTRACT

Loss of phylogenetic diversity (PD) has gained increasing attention in conservation biology. However, PD is not equally distributed in a phylogeny and can be better assessed when species relatedness (phylogenetic structure: PS) is also considered. Here, we investigate PD and PS in two refuges of biodiversity in northeastern Brazil: the Bahia Costal Forest (BCF) in the Atlantic Forest domain and Chapada Diamantina (CD) in the Caatinga domain. We used geographic data of 205 species at two spatial scales and a chronogram of Apocynaceae based on matK sequences to estimate PD and PS. Our results show an exceptionally high PD in both refuges, overdispersed in BCF and clustered in CD, although this difference is less evident or absent for recent relationships, especially at a smaller spatial scale. Overall, PS suggests long-term competitive exclusion under climatic stability, currently balanced by habitat filtering, in BCF, and biome conservatism and limited dispersal leading to in situ diversification and high density of microendemics in CD. The phylogenetically clustered flora in CD, also threatened by climate changes, are naturally more vulnerable than BCF. Therefore, while in situ conservation may ensure protection of biodiversity in BCF, emergency ex situ conservation is strongly recommended in CD.

1. INTRODUCTION Currently, the consensus is that biodiversity loss reduces community efficiency, stability, and productiveness [1], and the best strategy for biological conservation is through gains in phylogenetic diversity (PD) [2, 3]. Species do not contribute equally to total PD of an area but with their distinct evolutionary history [4, 5]. Closely related species, sharing a great extent of evolutionary history, are more likely to be redundant, whereas distantly related species are expected to play different ecological functions and provide different goods and services. Therefore, PD, based on the sum of branch length, is an important measure in conservation biology [5–7]. Species loss diminishes PD, but PD loss cannot be directly predicted by species loss, because proportions of PD loss may be higher than proportions of species loss when extinctions are clumped or biased to relictual lineages. Accordingly, communities whose species composition is phylogenetically clustered tend to lose evolutionary diversity more quickly, whereas communities in which species are phylogenetically overdispersed tend to lose less evolutionary diversity during extinctions [8, 9]. Therefore, PD loss depends on how communities are phylogenetically structured (species relatedness). Phylogenetic structure (PS) represents the overall relatedness in a species assembly (e.g., [10]) and combines community ecology and evolutionary thinking [11] into an interdisciplinary approach, community phylogenetics or ecophylogenetics [12]. PS is obtained by comparing the community phylogenetic distance to a null model, which randomize variants, such as species relationships and distributions [10, 11]. This metric differs from PD, which represents only the sum of phylogenetic distances of a community [5] and does not provide information on how species of this community are phylogenetically related. According to PS, communities may be phylogenetically clustered, which indicates co-occurrence of closely related species and suggests a stronger influence of an environmental filter on the community. In contrast, phylogenetically overdispersed communities indicate local exclusion of closely related species and suggest a stronger influence of interspecific competition and/or other density dependent negative interactions [11]. These interpretations are mainly supported by a strong tendency towards phylogenetic niche conservatism (PNC) during diversifications, as shown by Crisp et al. [13] for plants. According to PNC, closely related species tend to share traits and occupy similar habitats [14].However, PS is scale and context dependent, and alternative interpretations can emerge from similar patterns, often being equivocal when traits are not taken into account. For instance, clustered phylogenies can also result from character displacements among closely

7 related species allowing their coexistence or from limited dispersal and in situ speciation. On the other hand, overdispersed phylogenies may also result from convergent ecological traits among distantly related species. Finally, unstructured phylogenies suggest a balance between environmental constraints and biological interactions or a prevalent influence of neutral processes, such as a stochastic dynamic of dispersal, speciation, and extinction, rather than niche-based processes (e.g., [3, 11, 15– 19]). Furthermore, null models used for assessing PS can also affect results and eventually confer spurious structures for unstructured phylogenies (e.g., [16, 20–22]). Ecophylogenetics still lacks a consistent conceptual framework [12] and the use of PD as a proxy for functional diversity has been criticized for lacking empirical evidence [23]. However, PD and PS are complementary measures of biodiversity and can be properly used for biogeography, ecology, and conservation biology. Phylogenetics has been used to assess historical and ecological drivers at different spatial scales [12, 18], from latitudinal gradient of species richness (SR) [24], biogeographic processes during the Cenozoic [25], and coastal dune ecosystem [26] at a global scale to habitat heterogeneity [27, 28], successional pathways [29], and altitudinal gradient [30] at regional or, mainly, local scales. Although Brazil harbours the richest flora in the world, with more than 32,000 species of angiosperms [31], studies applying phylogeny to interpret plant composition (e.g., [32, 33]) are still scarce. Here, we investigate PD and PS in two centres of biodiversity (rich in species and endemisms), the Chapada Diamantina and the Bahia Costal Forest. Although only ∼120 km separates one from the other, these centres are under different environmental conditions and floristic domains and, together, comprise most of the angiosperm diversity in northeastern Brazil (Figure 1). Chapada Diamantina (CD) is the largest continuous plateau in the northern Espinhaço Range. Above 900m, particularly in the south and east, the plateau is covered by rocky fields (Campos Rupestres), an open vegetation biome associated with quartzite outcrops, rich in plant species and endemisms (e.g., [34]). It is floristically influenced by seasonally dry forests from the surrounding Caatinga but is also home of palaeomicroendemics [35] and was recently postulated as a historical refuge for fire-sensitive lineages [36]. The Bahia Costal Forest (BCF) ecoregion [37] is the largest area of climatic stability in the northern block of the Atlantic Forest [38–40]. Ranging from northern Espirito Santo to southern Bahia, it is dominated by evergreen forests, harbouring a forest refuge, with high levels of endemism [41] and one of the highest tree species densities in the world [42]. Since CD and BCF are species rich, high PD values are expected for both. However, more species of shrubs and

Figure 1. Northeastern Brazil in South America, showing the number of Apocynaceae species per region in a Venn diagram.

9 lianas are found in CD whereas BCF is richer in species of trees. This difference probably affected species relatedness and community resilience. Therefore, assessing PS in CD and BCF may provide important basis for phyloconservation policies to protect the high biodiversity found in these areas. For assessing PS in CD and BCF, we used Apocynaceae and ecophylogenetics in a macroecological approach. The Apocynaceae are one of the ten largest families of angiosperms and their SR distribution shows the highest correlation with angiosperm SR distribution in Brazil, when the five richest angiosperm families in the country are taken into account (Figure 2). This high correlation strongly confirms the Apocynaceae to be a good indicator for plant diversity across the Brazilian territory. The Apocynaceae consist of approximately 5,000 species [43] and 360 genera [44] and are also well represented in Brazil, with 770 species and 73 genera [45]. They are latescent plants with pentamerous, gamopetalous, isostemonous, bicarpelar flowers and comprise a broad range of habits (trees, shrubs, herbs, and lianas) and have pollen transferred as monads, tetrads, or in pollinia, berry- like or bifollicular fruits and seeds with or without coma. The family is widespread over the world, especially in tropical and subtropical regions, and occurs in almost any habitat, from lowland wet forests to deserts and grasslands in high altitudes [46, 47]. In Brazil, such diversity is classified in three subfamilies: Apocynoideae, Asclepiadoideae, and Rauvolfioideae [47]. Asclepiadoideae comprises a clade that consists mainly of shrubs and lianas,and CD is an important centre of diversity of the subfamily. Together, Apocynoideae and Rauvolfioideae comprise the early diverging lineages of Apocynaceae; the former consists mainly of shrubs and lianas, whereas trees are prevalent in the latter subfamily. Apocynoideae and Rauvolfioideae form a basal grade in Apocynaceae and their species are usually broadly distributed or inhabit predominantly tropical forests such as BCF. In this study, we map the Apocynaceae PD in northeastern Brazil to evaluate whether CD and BCF present exceptionally high PD and compare the Apocynaceae PS in the two areas. Taking into account the ecological specificities across the Apocynaceae phylogeny, we would expect to find clustered communities in both CD and BCF but concentrated in different lineages. The most derived Asclepiadoideae would be concentrated in CD whereas lineages of the Rauvolfioideae-Apocynoideae basal grade would be mainly concentrated in BCF. We then try to identify ecological and evolutionary factors that may have affected plant community in the two areas and suggest general perspectives for conserving the biodiversity in both.

Figure 2. Family species number × angiosperm species number per vegetation, states, and regions of Brazil. Spearman‘s correlation values for Apocynaceae and the five richest families of angiosperm in Brazil [31]: Apocynaceae, 푟 푠 = 0.98 (푃 < 2.2푒 − 16); Fabaceae, 푟 푠 = 0.95 (푃 < 2.2푒 − 16); Rubiaceae, 푟 푠 = 0.88 (푃 < 2.2푒 − 16); Orchidaceae, 푟 푠 = 0.81 (푃 = 5.47푒 − 15); Asteraceae, 푟 푠 = 0.71 (푃 = 3.29푒 − 10); and Poaceae, 푟 푠 = 0.28 (푃 = 0.03).

2. MATERIAL AND METHODS

We built a database with approximately 7,000 specimens, representing 205 species and 47 genera of Apocynaceae native to the Caatinga and Atlantic Forest domains in northeast Brazil (Appendix 1) based on exsiccates from the main herbaria in Brazil, Europe, and the United States. GPS coordinates were extracted from labels and confirmed or recovered with the help of Google Earth. Specimens without locality were treated at the municipal headquarter. A calibrated phylogeny was constructed with matK sequences of 142 species of Apocynaceae and five from the Loganiaceae (outgroup) from Genbank (Appendix 2). We sampled 95 genera, representing the five subfamilies and most tribes of Apocynaceae. Sequences were initially aligned in Muscle [48] and subsequently manually adjusted in

11 mesquite [49]. Age estimates were obtained using BEAST 1.8 [50] as implemented in CIPRES [51], using a GTR substitution model, gamma distribution, and relaxed molecular clock. The analysis was conducted from a random starting tree, with a Yule speciation model. Dating was calibrated using two fossils: a comose seed (Apocynospermum) from the Eocene (mean = 1.5, Std. Dev. = 1, and Offset = 47, Lognormal prior) assigned to the APSA clade stem node (Apocynoideae, Periplocoideae, Secamonoideae, and Asclepiadoideae) [52] and a tetrad (Polyporotetradites laevigatus) from the Oligocene/Miocene boundary (mean = 1.5, Std.Dev. = 1, and Offset = 23, Lognormal prior) assigned to Tacazzea (Periplocoideae stem node) [53]. A Monte Carlo-Markov chain was run for 5 × 107 generations, saving a tree every 2,000 generations. The log file was analysed in TRACER 1.6 [54] to assess whether the effective sample size reached 200 for all parameters. The maximum credibility tree was recovered in TreeAnnotator 1.8.0 [54], after deleting the first 10% of saved trees (burn-in). We assessed Apocynaceae PD and PS using a pseudochronogram constructed from the calibrated tree, in which species branch length was treated as an average of total lineage branch length. We estimated the total branch length of a lineage assuming half-aged, successive, and balanced dichotomies. Accordingly, species were added regularly at the mid distance of the longest branch of that lineage and the sum of lineage branch lengths was divided by the number of species of the lineage in northeastern Brazil (Figure 3). Eight genera lacking molecular data were included based on their taxonomic position and/or other molecular markers [47]. Since biodiversity metrics may be strongly affected by scales, we calculated SR, PD, and PS for 0.5°× 0.5° and 0.08°× 0.08° grids to test the consistence of observed patterns under different spatial scales. PD, after standardization of species branch length, and SR were calculated using Biodiverse v. 0.19 [55]. We estimated PS through net relatedness index (NRI) and nearest taxon index (NTI) for CD and BCF based on cells with exceptionally high PD (higher than 95% of cells for 05°× 05° grid and higher than 97.5% for 0.08°× 0.08° grid) using Phylocom 4.2 [56]. Null communities were generated adopting a model that randomizes species relationships keeping their original species richness [22, 57]. Species abundance was not considered because our data is based on herbarium material and analysed at relatively large scales. Ten thousand random (phylogenetically unstructured) communities were generated using the Apocynaceae pseudochronogram and a species pool from northeastern Brazil. This region is much larger than both CD and BCF, but only approximately one quarter of the species in northeastern Brazil are not represented in either CD or BCF (Figure 1). To assess statistical differences in SR, PD, NRI, and NTI between CD

12 and BCF, we used Kruskal-Wallis test and pairwise correlations between measures of diversity using Spearman‘s index, both in R statistical software [58]. To ensure that results of statistical tests are not overestimated due to spatial autocorrelation of analysis [59, 60], we also performed Kruskal-Wallis tests for SR, PD, and PS values without neighborhood cells at both spatial scales.

Figure 3. Procedure for calculating species‘ branch lengths. (a) Chronogram from Bayesian analysis. (b) Estimating lineage branch length: species lacking molecular data are inserted sequentially at the half length of the longest branches (dashed, orange branches). (c) Total branch length of the clade is standardized and species polytomized at the stem, which corresponds to the original age of lineage minus the species standardized branch length.

3. RESULTS

Apocynaceae chronogram from matK sequences (Figure S1; see Supplementary Material available online at http://dx.doi.org/10.1155/2015/758019) mostly agree with the topology summarized in Rapini [47], based on several phylogenetic studies, and also with the most recent classification of the family [44]. The few hard conflicts (posterior probability = 100%) were (1) Tylophora sister to Asclepiadinae - Cynanchinae clade, rather than within Cynanchinae; (2) Macropharynx, Peltastes, and Temnadenia forming a clade without Prestonia, but agreeing with Peltastinae circumscription [44]; and (3) Ambelania sister to Tabernaemontana, making Ambelaniinae paraphyletic. The origin of Apocynaceae (stem group) was estimated at 68.56 million years ago (Ma; HPD 95% = 55.92–84.4Ma), of APSA clade at 50.52Ma (47.19–56.38), and of Asclepiadoideae at 27.35Ma (21.57–33.48). Genera estimates ranged from36.83Ma (22.6– 52.9) for Rauvolfia to 4.8Ma (1.2–9.4) for the monotypic Hancornia (H. speciosa). The Atlantic Forest domain shelters 130 species of Apocynaceae in northeastern Brazil and the Caatinga domain 154; 79 of these species occur in both domains. PD, estimated from the pseudochronogram (Figure S2), and SR were strongly correlated (푃 ≤ 2.2푒 − 16, 푟 푠= 0.945) and showed similar distributions in northeastern Brazil, with exceptionally high values concentrated in the CD and BCF (Figure 4). CD corresponds to only 4.5% of Caatinga in northeastern Brazil but shelters 77% of its SR (118 of 154 species) and 78% of its PD; 22% (37 species) of SR from CD was not found elsewhere in Caatinga, representing 8% of a restricted PD. BCF corresponds to 41% of the northeastern Atlantic Forest and comprises 80%of its SR (103 of 130 species) and 88% of its PD; 44% (46 species) of SR from BCF is not found anywhere else in the northern Atlantic Forest, representing 21% of a restricted PD (Figure 1). PDs in CD and BCF are not statistically different, but their SR and NRIs are (Table 1). Overall, the Apocynaceae are phylogenetically clustered (NRI > 0) in CD and overdispersed (NRI < 0) in BCF. Recent relationships are not evidently structured in either region and the difference between them is not significant at small spatial scale (0.08° × 0.08°) when neighbour cells are not considered; otherwise, the difference is significant (Table 1), with CD appearing to be clustered (NTI > 0) only at a large spatial scale (0.5°× 0.5°) when neighbour cells are not considered and BCF tending toward overdispersion (NTI < 0) (Figure 5). SR correlates to PD but only correlates to NRI when 0.08° cells is used in

CD. SR is not statistically correlated to NTI and PD is not correlated to NRI or NTI in most cases (Table 2).

Table 2. Kruskal-Wallis test for species richness (SR), phylogenetic diversity (PD), net relatedness index (NRI), and nearest taxon index (NTI) in cells with exceptionally high PD in Chapada Diamantina and Bahia Costal Forest, using 0.5° × 0.5° and 0.08° × 0.08° cells, with (Nc) and without (wNc) one neighborhood cell.

0.5° Nc 0.5° wNc 0.08° Nc 0.08° wNc

p x² p x² p x² p x²

PD 0.6256 0.2381 0.2976 1.0848 0.5311 0.3923 0.2744 1.1946 SR 0.001 10.7407 0.03136 4.6333 3.49E-05 17.1326 0.0002 13.3278 NRI 0.0006 11.6667 0.001194 10.5 7.43E-07 24.5 2.96E-05 17.4414 NTI 0.005 7.8107 0.007774 7.0848 0.005194 7.8107 0.08184 3.028

Table 3. Spearman‘s correlation values for species richness (SR), phylogenetic diversity (PD), net relatedness index (NRI), and nearest taxon index (NTI) in cells with exceptionally high PDin the Bahia Costal Forest (BCF) and Chapada Diamantina (CD), using 0.5° × 0.5° and 0.08° × 0.08° cells, with (Nc) and without (wNc) one neighborhood cell; asterisks indicate the significance values (푃 < 0.05).

Scales SR-PD SR-NRI SR-NTI PD-NRI PD-NTI p-value rho p-value rho p-value rho p-value rho p-value rho 0.5° Nc 0.0003 0.9 0.91 0.03 0.47 0.25 0.82 -0.07 0.72 0.12 BCF 0.5° wNc 0.005 0.86 0.79 -0.1 0.12 -0.59 0.38 -0.35 0.028 -0.76 0.08° Nc 0.01 0.62 0.15 0.4 0.7 -0.11 0.28 -0.3 0.74 0.09 0.08° wNc 0.019 0.57 0.37 0.23 0.17 0.35 0.31 -0.26 0.38 -0.23 0.5° Nc 0.002519 0.92 0.7599 0.14 0.75 -0.14 0.87 -0.07 0.75 -0.14 0.5° wNc 0.01 0.85 0.21 0.53 0.75 -0.14 0.38 0.39 0.39 -0.39 CD 0.08° Nc 3.50E-09 0.92 0.0007 0.67 0.64 -0.1 5.89E-05 0.76 0.07 -0.39 0.08° wNc 0.0002 0.78 0.002 0.7 0.74 0.09 0.18 0.35 0.12 -0.39

Figure 4. Distribution of species richness (a) and phylogenetic diversity (b) of Apocynaceae in northeastern Brazil, using 0.5° ×0.5° cells with one neighborhood cell; 0.5° cells with black margins present exceptionally high (5% highest) phylogenetic diversity without neighborhood in Chapada Diamantina (CD) and Bahia Costal Forest (BCF); 0.08° cells present exceptionally high (2.5% highest) phylogenetic diversity with (black margins) and without neighborhood cells (black squares) in CD and BCF.

4. DISCUSSION

The Apocynaceae have been phylogenetically investigated using several molecular regions (summarized in [47]) and, more recently, plastome analyses were also employed to resolve major relationships in the APSA clade [61]. Advances in Apocynaceae systematics is incorporated into an updated classification at the tribal and subtribal levels [44], but many genera are not monophyletic (e.g., [62, 63]) or still need a thorough phylogenetic investigation [47]. So far, dated phylogenies in Apocynaceae focused only on less inclusive groups, such as Asclepiadoideae [64], Tylophorinae [65], and Minaria [66], and used trnL

Figure 5. Distribution of net relatedness index (NRI) and nearest taxon index (NTI) in northeastern Brazil with bloxplots comparing 32 cells with exceptionally high phylogenetic diversity (Figure 4) in Chapada Diamantina (CD) and Bahia Costal Forest (BCF), using different scales (0.5° × 0.5° and 0.08° × 0.08° cells), with (Nc) and without one neighborhood cell. intron and trnL-F intergenic space (trnL-F) for the Apocynaceae big picture. Therefore, this is the first Apocynaceae dated phylogeny using matK. Our results consistently recovered relationships obtained in previous studies and age estimates overlap those with trnL-F [66] when confidence intervals are taken into account. However, the small sampling in major groups of Apocynaceae prevents a comprehensive biogeographic discussion. Most of the Apocynaceae diversity in northeastern Brazil is concentrated in the Campos Rupestres of Chapada Diamantina (CD) and in the Bahia Costal Forest (BCF). CD

17 and BCF are historical refuges for plants in two different floristic domains. CD is considered a refuge for grasslands during interglacial periods (e.g., [34, 67, 68]) or for fire sensitive lineages after the expansion of the fire-prone Cerrado in Central Brazil [36, 66], whereas BCF is a refuge for forest associated lineages [40]. These plant refuges shelter an exceptionally high PD, which is phylogenetically clustered in CD but overdispersed in BCF. This difference is mirrored in SR, which is higher and spatially concentrated in CD. The Asclepiadoideae and the upper Apocynoideae grade are better represented in CD, contributing to a high number of young lineages,while the Apocynaceae basal grade, including Rauvolfioideae and the early Apocynoideae, is better represented in BCF, contributing to fewer but older lineages (Figure 6). Plants in tropical forests have usually presented clustered phylogenies (e.g., [10, 20, 69–71]), contrasting with BCF. There are several potential explanations for this difference. First, most studies in tropical forests considered only tree communities, which tend to be phylogenetically clustered [30]. Second, their phylogenetic scale often comprises the whole angiosperms, and scales that are phylogenetically more inclusive are more likely to produce clustered PS [15–17] and also lose the power for predicting ecological processes as convergent traits increase to deeper relationships [3]. Third, such analyses are usually produced from poorly resolved phylogenies, in which species are unresolved within genera and genera within families. A lack of phylogenetic resolution also limits the analysis power and results can be incorrect, particularly near the tips, because many families are still awaiting phylogenetic analyses at the genus level. In BCF, the overdispersed phylogeny at family scale is possibly produced by phenotypic repulsion caused by longterm competitive exclusion in a climatically stable region, rather than by phylogenetic attraction because of convergent traits, which is more likely at higher phylogenetic scales. When species are phylogenetically evenly distributed, as in BCF, niche overlap is expected to be reduced, and species probably have complementary fluctuations, responding differently to environmental changes and replacing one another in dominance, while maintaining ecosystem function. Because of that, diverse communities are more robust to species invasion, more productive and more resilient to environmental changes [12]. Under climatic changes, estimates are that in 50 years, neotropical ever-wet zones will be one-third smaller because of increasing seasonal variability in rainfall [72]. As such, refuges of biodiversity like BCF—stable climatic region that is home of a high and phylogenetically evenly distributed evolutionary diversity— deserve high priority for in situ conservation.

Figure 6. Backbone of Apocynaceae pseudochronogram (modified from Figure S2) showing the lineages represented in the exceptionally high PD cells of the Chapada Diamantina (left) and Bahia Costal Forest (right) in bold.

The Apocynaceae are spatially and phylogenetically compact in CD, a pattern different from that in BFC. The overall clustering in CD reflects the heterogeneity and fragmented distribution of Campos Rupestres. At mountaintops, this biome consists of a mosaic of microhabitats at a small scale with an insular distribution at a large scale, resulting in high 훽 diversity [73]. Accordingly, the same spatial scales used in BCF tend to comprise more heterogeneous areas in CD and, therefore, are more likely to support species with different ecological traits in larger clades (e.g., Metastelmatinae) and also a higher number of allopatric, closely related, microendemic species with similar ecological traits. This biogeographic pattern is reflected by the overall clustered PS in CD. For recent relationships, the phylogeny is not evidently structured, suggesting a stronger influence of neutral processes. Therefore, at a biogeographic context, deeper and narrower phylogenetic structures together suggest a stronger influence of niche conservatism, limited dispersal and in situ diversification in CD, which can explain the high density of microendemic species in the Campos Rupestres as a result of nonadaptive, geographic radiations, as postulated by Ribeiro et al. ([36]; see also [66]). According to the most popular hypothesis of diversification in the Espinhaço Range, the Campos Rupestres is cold associated, contracting to highlands during warmer periods and

19 expanding to lowlands during cooler periods. Diversification resulted from successive contraction-expansion cycles caused by Pleistocene climatic fluctuations (e.g., [34, 67, 68]), as also suggested by refugial sites in different continents (e.g., [74, 75]). Alternatively, highlands represent refuges for fire-sensitive lineages and diversification was driven by the expansion of fire-prone Cerrado and fragmentation of Campos Rupestres since the late Miocene-Pliocene [36]. According to this hypothesis, milder weather and a high concentration of rocky outcrops in the highlands have helped to prevent frequent, intense fires, and diversification is result of a long-term contraction of the Campos Rupestres. Both scenarios consider mountaintops along the Espinhaço Range ecologically stable areas buffering biome conservative lineages during environmental changes and fit the PS recovered for Apocynaceae here. Phylogenetic diversity and species relatedness reflect important properties for community function and stability [12, 76]. The high PD in CD resulted from high SR of closely related species, in clustered structured communities; therefore, communities in CD are probably more vulnerable than in BCF. Past and future distribution models estimated a smaller distribution of Campos Rupestres today compared to in the Last Maximum Glacial and even smaller distributions in the future, almost disappearing in CD by the end of this century because of increasing seasonality [77]. Under this scenario, the reduction of the Campos Rupestres range is a natural process and loss of biodiversity in CD is probably inevitable. However, this process has been greatly accelerated by anthropogenic changes and CD may not represent a biodiversity refuge in the short future. Therefore, ex situ conservation is probably an important strategy to retain the biodiversity from the Campos Rupestres of the CD available in the future.

5. CONCLUSION

Ecology and phylogeny have been used independently from each other in biogeography [24]. Ecophylogenetics fills part of this gap and its use for investigating community structure has been increasing quickly [78]. However, ecophylogenetics and macroecology are still somewhat separate from each other, and few authors (e.g., [25, 79]) dared to analyse PS at large geographical scales. Although attention has been given more recently to species evolutionary distinctiveness, different components of a multifaceted biodiversity cannot be confidently used as surrogate of others (e.g., [80–82]).

The use of phylogenies through PD (sensu Faith [5]) in conservation goes beyond the traditional SR because it also includes evolutionary information. However, PD does not take into account species relationships and therefore misses an important aspect from which processes and factors that have driven community diversity can be inferred and functional diversity can be estimated [80]. PS emerges as a key concept in this context because it distributes PD across SR, shaping the evolutionary trace of a community, and, at the same time, provides indirect access for functional diversity, in particular when traits show a high phylogenetic signal. Thus, PS becomes an important measure to assess the vulnerability of communities against climatic changes or ecological and anthropogenic disturbances, providing information for conservation, and can be used for both understanding the past and anticipating the future. SR, PD, and PS represent different aspects of biodiversity, and, together, they provide a more complete framework for conservation assessments. Our study shows that individual extinctions are probably more influent phylogenetically in BCF than in CD because PD/SR ratio is higher in BCF. However, communities in BCF are usually overdispersed and, therefore, probably more resilient against invasive species, climatic changes, and anthropogenic disturbances than communities in CD, which are phylogenetically unstructured or more often clustered. Based on PS pattern, different general conservation strategies can be designed; while in situ conservation may fit well for communities in BCF, an ex situ conservation is also recommended for protecting biodiversity in CD.

ACKNOWLEDGEMENTS

This study is part of the M.Sc. thesis of LP, which was developed at PPGBot-UEFS with a fellowship from CNPq. AR is supported by Pq-1D CNPq grant. We also thank CNPq for supporting the project on ―Phyloconservation‖ (no. 485468/2013-1)

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SUPPLEMENTARY FIGURES

SUPPLEMENTARY FIGURE 1

Figure S1. Apocynaceae chronogram, highlighting lineages represented in northeastern Brazil (bold), including estimated ages (Ma = million years ago) and the 95% credibility interval (HPD) in the table. Posterior probabilities: * = 90–94%, ** = 95–99%, *** = 100%.

SUPPLEMENTARY FIGURE 2

Figure S2. Apocynaceae pseudochronogram (modified from chronogram in Figure S1 according to procedure illustrated in Figure 3), showing branch length and clades used for estimating the phylogenetic structure in northeastern Brazil. Triangle sizes are vertically proportional to the number of species in northeastern Brazil and horizontally proportional to the branch lengths used for species of that clade.

APPENDICES

APPENDIX 1

Appendix 1. Native species of Apocynaceae from northeastern Brazil (Caatinga and Atlantic Forest domains in the Northeast Brazil), indicating species occurring in the Caatinga and Atlantic forest. CD denotes species occurring in Chapada Diamantina and BCF in Bahia Costal Forest, in bold when they are endemic to these regions and with an asterisk when they appear in 0.08° × 0.08° cells with exceptionally high phylogenetic diversity, considering one neighborhood cell.

Floristic Domains Species Caatinga Atlantic Forest Allamanda blanchetii A.DC. CD* Allamanda calcicola Souza-Silva & Rapini x Allamanda cathartica L. BCF* Allamanda doniana Müll.Arg. x BCF* Allamanda martii Müll.Arg. BCF* Allamanda puberula A.DC. CD* BCF Allamanda thevetifolia Müll.Arg. x Araujia sericifera Brot. x Asclepias candida Vell. CD* Asclepias curassavica L. CD* BCF* Asclepias mellodora A.St.-Hil. CD* BCF* Aspidosperma cuspa S.F.Blake ex Pittier CD Aspidosperma cylindrocarpon Müll.Arg. x Aspidosperma discolor A.DC. CD* BCF* Aspidosperma illustre (Vell.) Kuhlm. & Pirajá x BCF Aspidosperma limae Woodson x Aspidosperma macrocarpon Mart. CD Aspidosperma multiflorum A.DC. x x Aspidosperma parvifolium A.DC. CD* BCF* Aspidosperma polyneuron Müll.Arg. CD* BCF Aspidosperma pyricollum Müll.Arg. x Aspidosperma pyrifolium Mart. CD* BCF* Aspidosperma ramiflorum Müll.Arg. BCF Aspidosperma schultesii Woodson BCF Aspidosperma spruceanum Benth. ex Müll.Arg. CD* BCF* Aspidosperma subincanum Mart. x Aspidosperma thomasii Marc.-Ferr. BCF* Aspidosperma tomentosum Mart. CD* x

Floristic Domains Species Caatinga Atlantic Forest Aspidosperma ulei Markgr. x Bahiella blanchetii (A.DC.) J.F.Morales BCF* Bahiella infundibuliflora J.F.Morales BCF Barjonia chlorifolia Decne. CD* Barjonia erecta (Vell.) K.Schum. CD* Barjonia glazioui Marquete CD* Blepharodon ampliflorum E.Fourn. CD* x Blepharodon bicolor Decne. CD Blepharodon costae Fontella & Morillo BCF * Blepharodon manicatum (Decne.) Fontella CD* Blepharodon pictum (Vahl) W.D.Stevens CD* BCF* Condylocarpon intermedium Müll.Arg. BCF* Condylocarpon isthmicum (Vell.) A.DC. CD* BCF* Couma rigida Müll.Arg. CD* BCF* Cynanchum montevidense Spreng. x x Cynanchum roulinioides (E.Fourn.) Rapini CD* Ditassa arianeae Fontella & E.A.Schwarz x BCF* Ditassa blanchetii Decne. BCF* Ditassa capillaris E.Fourn. CD* BCF* Ditassa congesta E.Fourn. x Ditassa crassifolia Decne. CD BCF* Ditassa dardanoi T.U.P.Konno & Wand. CD Ditassa dolichoglossa Schltr. x Ditassa glazioui E.Fourn. CD* x Ditassa grandiflora E.Fourn. CD* Ditassa hastata Decne. CD* x Ditassa hispida (Vell.) Fontella CD BCF* Ditassa lenheirensis Silveira CD* x Ditassa melantha Silveira CD* Ditassa obcordata Mart. CD* Ditassa oxyphylla Turcz. CD* BCF Ditassa pohliana E.Fourn. CD* BCF Ditassa retusa Mart. CD* x Ditassa rotundifolia (Decne.) Baill. ex K.Schum. CD* x Ditassa succedanea Rapini CD* Fischeria stellata (Vell.) E.Fourn. CD BCF Forsteronia australis Müll.Arg. CD* BCF Forsteronia glabrescens Müll.Arg. x Forsteronia leptocarpa (Hook. & Arn.) A.DC. CD BCF* Forsteronia montana Müll.Arg. BCF* Forsteronia pubescens A.DC. CD* x Forsteronia rufa Müll.Arg. CD* BCF Forsteronia thyrsoidea Müll.Arg. CD* BCF

Floristic Domains Species Caatinga Atlantic Forest Funastrum clausum (Jacq.) Schltr. CD* BCF* Geissospermum laeve (Vell.) Miers BCF* Gonolobus parviflorus Decne. x BCF* Gonolobus rostratus (Vahl) R.Br. ex Shult. x Hancornia speciosa Gomes CD* BCF* Hemipogon carassensis (Malme) Rapini CD* Himatanthus bracteatus (A. DC.) Woodson CD* BCF* Himatanthus drasticus (Mart.) Plumel CD* BCF Himatanthus obovatus (Müll. Arg.) Woodson CD BCF* Himatanthus phagedaenicus (Mart.) Woodson x Jobinia connivens (Hook. & Arn.) Malme CD* Jobinia lindbergii E.Fourn. CD* Lacmellea bahiensis J.F.Morales BCF* Lacmellea pauciflora (Kuhlm.) Markgr. BCF Macoubea guianensis Aubl. BCF* Macroditassa laurifolia (Decne.) Fontella CD* BCF* Malouetia cestroides (Nees ex Mart.) Müll.Arg. BCF* Mandevilla alexicaca (Mart. ex Stadelm.) M.F.Sales CD* Mandevilla bahiensis (Woodson) M.F.Sales & Kin.-Gouv. CD* BCF* Mandevilla catimbauensis Souza-Silva et al. x Mandevilla dardanoi M.F.Sales et al. x x Mandevilla emarginata (Vell.) C.Ezcurra CD* Mandevilla fistulosa M.F.Sales et al. BCF Mandevilla guanabarica Casar. ex M.F.Sales, Kin.-Gouv. & A.O.Simões BCF Mandevilla hatschbachii M.F.Sales et al. CD Mandevilla hirsuta (A.Rich.) K.Schum. CD* BCF Mandevilla illustris (Vell.) Woodson CD* x Mandevilla leptophylla (A.DC.) K.Schum. CD* Mandevilla longiflora (Desf.) Pichon CD* Mandevilla luetzelburgii Woodson CD BCF Mandevilla martiana (Stadelm.) Woodson CD* Mandevilla martii (Müll.Arg.) Pichon CD Mandevilla microphylla (Stadelm.) M.F.Sales & Kin.-Gouv. CD* BCF* Mandevilla moricandiana (A.DC.) Woodson CD* BCF* Mandevilla myriophylla (Taub. ex Ule) Woodson CD Mandevilla permixta Woodson BCF* Mandevilla sancta (Stadelm.) Woodson CD* x Mandevilla scabra (Hoffmanns. ex Roem. & Schult.) K.Schum. CD* BCF* Mandevilla tenuifolia (J.C.Mikan) Woodson CD* x Marsdenia altissima (Jacq.) Dugand CD* BCF Marsdenia caatingae Morillo x BCF* Marsdenia carvalhoi Morillo & Carnevali BCF

Floristic Domains Species Caatinga Atlantic Forest Marsdenia dorothyae Fontella & Morillo BCF* Marsdenia heringeri Fontella x Marsdenia hilariana E.Fourn. CD* BCF Marsdenia loniceroides E.Fourn. x x Marsdenia macrophylla (Humb. & Bonpl. ex Schult.) E.Fourn. x Marsdenia megalantha Goyder & Morillo x Marsdenia pickelii Fontella & Morillo x Marsdenia queirozii Fontella x Marsdenia suberosa (E.Fourn.) Malme CD* BCF Marsdenia zehntneri Fontella CD Matelea bahiensis Morillo & Fontella BCF* Matelea denticulata (Vahl) Fontella & E.A.Schwarz x BCF* Matelea endressiae Fontella & Goes CD Matelea ganglinosa (Vell.) Rapini CD* BCF* Matelea harleyi Fontella & Morillo CD Matelea morilloana Fontella CD* Matelea nigra (Decne.) Morillo & Fontella CD* Matelea orthosioides (E.Fourn.) Fontella CD* BCF* Matelea pedalis (E.Fourn.) Fontella & E.A.Schwarz CD* Matelea riparia Morillo BCF Matelea roulinioides Agra & W.D.Stevens x Matelea santosii Morillo & Fontella BCF Metastelma giuliettianum Fontella CD* Metastelma harleyi Fontella CD* Metastelma myrtifolium Decne. CD* Minaria acerosa (Mart.) T.U.P.Konno & Rapini CD* Minaria cordata (Turcz.) T.U.P.Konno & Rapini CD* x Minaria decussata (Mart.) T.U.P.Konno & Rapini CD Minaria harleyi (Fontella & Marquete) Rapini & U.C.S.Silva CD* Minaria volubilis Rapini & U.C.S.Silva CD* Monsanima morrenioides (Goyder) Liede & Meve CD* Nephradenia asparagoides (Decne.) E.Fourn. CD* Odontadenia hypoglauca Müll.Arg. x Odontadenia lutea (Vell.) Markgr. CD* BCF* Orthosia parviflora (E.Fourn.) Liede & Meve BCF* Orthosia scoparia (Nutt.) Liede & Meve x Oxypetalum arachnoideum E.Fourn. CD* Oxypetalum banksii R.Br. ex Schult. CD BCF* Oxypetalum capitatum Mart. CD* Oxypetalum erostre E.Fourn. CD* Oxypetalum harleyi (Fontella & Goyder) Farinaccio CD* BCF Oxypetalum jacobinae Decne. CD* BCF

Floristic Domains Species Caatinga Atlantic Forest Oxypetalum laciniatum Rapini & Farinaccio x Oxypetalum montanum Mart. CD* Oxypetalum pachyglossum Decne. BCF* Oxypetalum strictum Mart. CD* Oxypetalum warmingii (E.Fourn.) Fontella & Marquete x Peltastes peltatus (Vell.) Woodson BCF* Peltastes pulcher (Miers) J.F.Morales BCF* Peplonia adnata (E.Fourn.) U.C.S.Silva & Rapini CD* BCF Peplonia asteria (Vell.) Fontella & E.A.Schwarz BCF* Peplonia axillaris (Vell.) Fontella & Rapini BCF Peplonia bradeana (Fontella & E.A.Schwarz) Fontella & Rapini BCF* Peplonia macrophylla (Malme) U.C.S.Silva & Rapini CD* Petalostelma cearense Malme x Petalostelma dardanoi Fontella x Petalostelma martianum (Decne.) E.Fourn. x Prestonia annularis G.Don BCF Prestonia bahiensis Müll.Arg. CD* BCF* Prestonia calycina Müll.Arg. x BCF Prestonia coalita (Vell.) Woodson CD* BCF* Prestonia didyma (Vell.) Woodson BCF* Prestonia erecta (Malme) J.F.Morales CD Prestonia lagoensis (Müll.Arg.) Woodson CD* Prestonia quinquangularis (Jacq.) Spreng. BCF* Rauvolfia atlantica Emygdio BCF* Rauvolfia bahiensis A.DC. BCF* Rauvolfia grandiflora Mart. x BCF* Rauvolfia ligustrina Willd. x x Rauvolfia mattfeldiana Markgr. CD Rauvolfia moricandii A.DC. BCF* Rauvolfia paucifolia A.DC. CD Schubertia grandiflora Mart. CD* Schubertia morilloana Fontella CD* Schubertia multiflora Mart. CD* BCF Secondatia densiflora A.DC. CD* x Secondatia floribunda A.DC. CD* BCF* Skytanthus hancorniifolius (A.DC.) Miers CD* x Stenomeria decalepis Turcz. BCF* Stipecoma peltigera (Stadelm.) Müll.Arg. CD* x Tabernaemontana catharinensis A.DC. x x Tabernaemontana flavicans Willd. ex Roem. & Schult. BCF* Tabernaemontana grandiflora L. BCF Tabernaemontana hystrix Steud. BCF Tabernaemontana laeta Mart. x BCF

Floristic Domains Species Caatinga Atlantic Forest Tabernaemontana salzmannii A.DC. BCF* Tabernaemontana solanifolia A.DC. CD* BCF Tassadia burchellii E.Fourn. CD* Tassadia obovata Decne. BCF* Tassadia propinqua Decne. BCF* Temnadenia odorifera (Vell.) J.F.Morales BCF* Temnadenia violacea (Vell.) Miers CD* BCF

Appendix 2. Taxa (Genbank accession number of matK sequence).

Taxa (Genbank Accession Number of matK Sequence)

These include the following: Allamanda cathartica L. (Z70190); Acokanthera oblongifolia Benth. & Hook.f. (Z70182); Allamanda schottii Pohl (DQ660495); Alstonia boonei De Wild. (KC627675); Alstonia scholaris (L.) R.Br. (AJ429321); Alyxia reinwardtii Blume (DQ660496); Alyxia oblongata Domin (EF456370); Ambelania acida Aubl. (DQ660497); Apocynum cannabinum L. (DQ660500); Apocynum androsaemifolium L. (Z70183); Araujia sericifera Brot. (Z98194); Asclepias syriaca L. (DQ660501); Asclepias curassavica L. (DQ026716); Aspidosperma cylindrocarpon M¨ull.Arg. (DQ660503); Aspidosperma australe M¨ull.Arg. (DQ660502); Baissea multiflora A.DC. (EF456319); Baissea zygodioides Stapf (DQ221120); Barjonia laxa Malme (JN805851); Barjonia cymosa E.Fourn. (JN805848); Barjonia erecta (Vell.) K.Schum. (JN805849); Blepharodon lineare (Decne.) Decne. (DQ026718); Blepharodon ampliflorum E.Fourn. (JN805852); Blepharodon nitidum (Vell.) J.F.Macbr. (DQ026720); Calotropis gigantea (L.) W.T.Aiton (JN228932); Carissa ovata R.Br. (DQ660506); Carissamacrocarpa A.DC. (DQ660505); Condylocarpon isthmicum (Vell.) A.DC. (DQ660511); Condylocarpon amazonicum (Markgr.) Ducke (DQ837537); Couma guianensis Aubl. (DQ660512); Cryptolepis sinensis Merr. (EF456374); Cryptostegia grandiflora R.Br. (Z98176); Cynanchum acutum L. (AY899939); Cynanchum auriculatum Buch.-Ham. ex Wight (GU373529); Ditassa banksii R.Br. ex Schult. (DQ026719); Ditassa auriflora Rapini (JN805856); Ditassa retusa Mart. (DQ026728); Dregea sinensis Hemsl. (Z98188); Dyera costulata Hook.f. (DQ660515); Farquharia elliptica Stapf (EF456357); Fockea capensis Endl. (Z98187); Fockea edulis K.Schum. (EF456383); Forsteronia velloziana (A.DC.) Woodson (DQ522596); Forsteronia acouci A.DC. (DQ522594); Funastrum clausum Schltr. (DQ026730); Galactophora schomburgkiana Woodson (EF456300); Geissospermum laeve Miers (DQ660517); Gomphocarpus fruticosus R.Br. (HM850833); Gonolobus xanthotrichus Brandegee (Z98195); Gymnanthera oblonga (Burm.f.) P.S.Green (EF456376); Hancornia speciosa Gomes (DQ660519); Hemipogon abietoides E.Fourn.(JN805868); Hemipogon luteus E.Fourn. (JN805874); Himatanthus bracteatus (A.DC.) Woodson (EF456366); Hunteria eburnea Pichon (DQ660521); Hunteria umbellate Hallier f. (KC627769); Hunteria zeylanica Gardner ex Thwaites (JX517717); Lacmellea aculeata (Ducke) Monach. (DQ660523); Lacmellea panamensis (Woodson) Markgr. (GQ982028); Logania vaginalis (Labill.) F.Muell. (AJ429324); Macoubea guianensis Aubl. (GU973901); Macroditassa grandiflora (E.Fourn.) Malme (JN805877); Macroditassa adnata Malme (JN805876); Macroditassa melantha (Silveira) Rapini (JN805879); Macropharynx renteriae A.H.Gentry (JQ586549); Macroscepis hirsuta (Vahl) Schltr. (JQ586771); Malouetia tamaquarina A.DC. (EF456346); Malouetia bequaertiana Woodson (EF456358); Mandevilla hirsuta (Rich.) K.Schum. (DQ522614); Mandevilla syrinx Woodson (DQ522637); Marsdenia engleriana W.Rothe (JQ586772); Melodinus australis (F.Muell.) Pierre (DQ660524); Melodinus cochinchinensis (Lour.) Merr. (DQ660525); Mesechites mansoanus (A.DC.) Woodson (DQ522644); Mesechites roseus (A.DC.) Miers (DQ522646); Metastelma schaffneri A.Gray (JN805884); Metastelma parviflorum (Sw.) Schult. (JN805883); Minaria harleyi (Fontella & Marquete) Rapini & U.C.S.Silva (JN805850); Minaria volubilis Rapini & U.C.S.Silva (JN805871); Minaria grazielae (Fontella & Marquete) T.U.P.Konno & Rapini (DQ026724); Mitreola petiolata (J.F.Gmel.) Torr. & A.Gray (JQ588153); Molongum laxum (Benth.) Pichon (Z70185); Mondia ecornuta (N.E.Br.) Bullock (AY899941);Motandra guineensisA.DC.

(DQ221121); Mucoa duckei (Markgr.) Zarucchi (GU973902); Nautonia nummularia Decne. (JN805886); Neocouma ternstroemiacea (Müll.Arg.) Pierre (GU973903); Nephradenia filipes Malme (JN805889); Nephradenia asparagoides E.Fourn (JN805888); Nephradenia acerosa Decne. (JN805887); Nerium oleander L. (Z98173); Odontadenia lutea (Vell.) Markgr. (DQ522648); Odontadenia perrottetii (A.DC.); Woodson (EF456272); Oncinotis tenuiloba Stapf (DQ660529); Orthosia scoparia (Nutt.) Liede & Meve (KF539851); Oxypetalum sublanatum Malme (DQ026738); Oxypetalum banksii Schult. (DQ026735); Parahancornia fasciculata (Poir.) Benoist (DQ660530); Peltastes isthmicus Woodson (EF456301); Peltastes peltatus (Vell.) Woodson (DQ660532); Pentopetia grevei (Baill.) Venter (AY899943); Peplonia macrophylla (Malme) U.C.S.Silva & Rapini (JN805878); Peplonia organensis E.Fourn.) Fontella & Rapini (JN805891); Peplonia asteria (Vell.) Fontella & E.A.Schwarz (JN805890); Periploca graeca L. (Z98178); Pervillaea phillipsonii Klack. (AJ312408); Pervillaea venenata (Baill.) Klack. (Z98181); Petalostelma martianum E.Fourn. (JN805892); Petalostelma sarcostemma (Lillo) Liede & Meve (JN805893); Petopentia natalensis (Schltr.) Bullock (DQ660533); Philibertia discolor (Schltr.) Goyder (DQ026732); Philibertia lysimachioides (Wedd.) T.Mey. (DQ026741); Phyllanthera grayi (P.I.Forst.) Venter (DQ660534); Pleiocarpa pycnantha Stapf (JX517964); Pleiocarpa mutica Benth. (DQ660535); Pleiocarpa rostrata Benth. (KC627551); Prestonia lagoensisWoodson (EF456329); Prestonia mexicana A.DC. (EF456345); Raphionacme flanaganii Schltr. (EF456377); Raphionacme welwitschii Schltr. & Rendle (Z98179); Rauvolfia vomitoria Wennberg (DQ660538); Rauvolfia mannii Stapf (Z70181); Rhabdadenia biflora Mull.Arg. (EF456277); Rhabdadenia macrostoma Mull.Arg. (EF456349); Rhigospira quadrangularis (Müll.Arg.) Miers (GU973904); Secamone elliptica R.Br. (DQ660541); Secamone bosseri Klack. (Z98182); Secamone geayi Costantin & Gallaud (Z98184); Secamonopsis madagascariensis Jum. (Z98185); Secondatia densiflora A.DC. (DQ522653); Spigelia anthelmia L. (JQ588156); Spongiosperma macrophyllum (Müll.Arg.) Zarucchi (GU973905); Stapelia gigantea N.E.Br. (JQ025000); Stephanostema stenocarpum K.Schum. (DQ660543); Stipecoma peltigera Müll.Arg. (EF456314); Strychnos axillaris Colebr. (AB636276); Tabernaemontana cuspidata Rusby (GU973931); Tabernaemontana hystrix Steud. (GU973942); Tacazzea apiculata Oliv. (AY899945); Temnadenia violacea (Vell.) Miers (EF456341); Temnadenia odorifera (Vell.) J.F.Morales (EF456353); Thevetia peruviana K.Schum. (Z70188); Thevetia ahouai A.DC. (GQ982112); Toxocarpus villosus Decne. (EF456379); Tylophora indica Merr. (Z98193); Vallesia antillana Woodson (AM295075); Wrightia dubia Spreng. (EF456257);Wrightia lanceolata Kerr (EF456291).